*Recombinant

Peptide vaccine

ANUJ KUMAR RAJAPhD. 1st yearAnimal Biotechnology centerNDRI, INDIA

*The agent stimulates the body's immune system to recognize

the agent as foreign, destroy it, and "remember" it, so that the

immune system can more easily recognize and destroy any of

these microorganisms that it later encounters.

VACCINE ???? *A vaccine is a biological preparation that

improves immunity to a particular disease.

A vaccine typically contains an agent that resembles a disease-causing microorganism

Weakened or killed forms of the microbe

its toxins Surface proteins

*

Live, attenuated vaccines

Inactivated vaccines

Subunit vaccines

Recombinant peptide vaccines

Conjugate vaccines

Toxoid vaccines

Recombinant live vector vaccines

Virus-like particles (VLPs)

“Naked” DNA vaccines

Edible vaccinesDendritic cell vaccines

*

Recombinant peptide vaccines consist of protein

antigens that have been produced in a heterologous

expression system (e.g., bacteria or yeast).

The vaccinated person produces antibodies to the

protein antigen, thus protecting him/her from

disease.

Hepatitis B Virus

Viral coat protein

surface antigen,

HBsAg

Highly immunogenic

particles have been

isolated from

infected persons

and used as a

vaccine.

Virus cannot be grown

in to a culture to

produce the protein.

Serious

limitationRisk of final

preparation being

contaminated

with active virion

and other type of

pathogens.

Constant

supply of

plasma from

infected

individuals

Therefore

efforts has

been made to

produce

HBsAg by

recombinant

means

Why there is a need to form recombinant peptide

vaccine ?

1

2

Procedure for development of recombinant peptide vaccine

Pathogenic

microorganismEpitope

cDNA

Expression

Vector

transfection

Selection of

recombinant

Production of

recombinant

peptides

Purification of

recombinant

peptide Purified peptide

VaccinationChecking for

immunogenecity

Presentation of peptides by MHC molecule

Example… Hepatitis E Virus

HEV has emerged as a significant cause of sporadic cases and extended outbreaks of acute hepatitis in many parts of the world.

The 7.5 kb single stranded positive sense RNA genome is predicted to contain 3 open reading frames (ORF).

ORF1 - Non-structural viral proteins

ORF 2 - major structural protein.

It is suggested that product of ORF 2 gene may be antigenic

determinants and raised the possibility of bacterially expressed

peptide as an HEV vaccine candidate.

The dimeric form of the peptide elicited a vigorous antibody

response in experimental animals and the resulting antisera

were found to cross-react against HEV, effecting an efficient

immune capture of the virus.

A 23 kDa peptide locating to amino acid residues 394 to 604 of

the major Hepatitis E Virus (HEV) structural protein was

expressed in E. coli.

Extraction of Viral RNA & cDNApreparation

Cloning of HEV sequence in pGEX expression vector

Production and purification of HEV peptides.

Immune capture of HEV

Reactivity of human sera against purified pE2

Steps involved in preparation of recombinant peptide vaccines

The viral genome was reverse transcribed using the primer E5R.

Vector for Expression of peptide

fragment

The cloned

sequence was

ligated to the

BamH1 and EcoR1

cloning sites on

the pGEX vector

and expressed as

GST fusion

peptide in E coli.

Bacterial cytosol

Recombinant plasmids were transferred into E. coli.

Transformants were selected as ampicillin resistant clones in LB agar.

Plasmid was extract from these transformants.

The cloned sequences were recovered by EcoRI and BamHIdigestion and their identity was confirmed by sequence analysis.

An overnight culture of the transformant was grown.

Bacterial cytosol containing the soluble fusion peptide was allowed to bind with glutathione conjugated sepharose 4B and the purified GST fusion peptide was eluted with glutathione.

Purified fusion protein was designated GE2.

Alternatively, the moiety of HEV peptide was obtained by thrombin cleavage.

This purified HEV peptide was denoted as pE2.

Production and purification of HEV peptides

*

*Polystyrene paddles were coated with rabbit anti-GE2 to capture

the HEV.

*Nested RT-PCR for detection of HEV RNA.

* The outer primer pair was A5F and A3R and the inner primer pair

was B5F and B3R (Table I).

Hyperimmune sera against GE2 were raised in rabbits.

The animals were given four bi-weekly intramuscular doses of the purified peptide.

The first dose was mixed with complete Freund's adjuvant, and subsequent doses were mixed with incomplete Freund's adjuvant.

The animals were bled on the 9th week.

*

Serially diluted rabbit pE2 antiserum was titrated by Western

blotting against an equal mixture of a heated and an unheated

sample of purified E2.

Limiting dilution of the serum reactive against the 42 kDa E2

dimer was 1:6,400 and that against the 23 kDa E2 monomer was

1:800.

Feasible even if virus cannot be cultivated

Requires primary course of injections followed by boosters.

Advantages

Production and quality control simpler

No other viral or external proteins, therefore less toxic.

Safer in cases where viruses are oncogenic or establish a

persistent infection.

Limitation

May be less immunogenic than conventional inactivated

whole-virus vaccines.

Requires adjuvant

*Hepatitis B. The vaccine uses hepatitis B

surface antigen produced in yeast.

*B subunit of cholera toxin.

*Vaccine against TB.

Examples of vaccine produced by Recombinant means.